The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration.

BACKGROUND: The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. METHODS: An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. RESULTS: Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. CONCLUSIONS: This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.

Solid-phase assays for study of carbohydrate specificity of galectins.

We have recently shown that the carbohydrate-binding pattern of galectins in cells differs from that determined in artificial (non-cellular) test-systems. To understand the observed discrepancy, we compared several test-systems differing in the mode of galectin presentation on solid phase. The most representative system was an assay where the binding of galectin (human galectins-1 and -3 were studied) to asialofetuin immobilized on solid phase was inhibited by polyacrylamide glycoconjugates, Glyc-PAA. This approach permits us to range quantitatively glycans (Glyc) by their affinity to galectin, i.e. to study both high and low affinity ligands. Our attempts to imitate the cell system by solid-phase assay were not successful. In the cell system galectin binds glycoconjugates by one carbohydrate-recognizing domain (CRD), and after that the binding to the remaining non-bound CRD is studied by means of fluorescein-labeled Glyc-PAA. In an "imitation" variant when galectins are loaded on adsorbed asialofetuin or Glyc-PAA followed by revealing of binding by the second Glyc-PAA, the interaction was not observed or glycans were ordered poorly, unlike in the inhibitory assay. When galectins were adsorbed on corresponding antibodies (when all CRDs were free for recognition by carbohydrate), a good concentration dependence was observed and patterns of specificities were similar (though not identical) for the two methods; notably, this system does not reflect the situation in the cell. Besides the above-mentioned, other variants of solid-phase analysis of galectin specificity were tested. The results elucidate the mechanism and consequence of galectin CRD cis-masking on cell surface.

Design of the blood group AB glycotope.

Although the nature of the blood groups A and B has been comprehensively studied for a long time, it is still unclear as to what exactly is the epitope that is recognized by antibodies having AB specificity, i.e. monoclonal and polyclonal antibodies which are capable of interacting equally well with the antigens GalNAcalpha 1-3(Fucalpha 1-2)Gal (A trisaccharide) and Galalpha 1-3(Fucalpha 1-2)Gal (B trisaccharide), but do not react with their common fragment Fucalpha 1-2Gal. We have supposed that besides Fucalpha 1-2Gal, A and B antigens have one more shared epitope. The trisaccharides A and B are practically identical from the conformational point of view, the only difference being situated at position 2 of Galalpha residue, i.e. trisaccharide A has a NHAc group, whereas trisaccharide B has a hydroxyl group (see formulas). We have hypothesized that the AB-epitope should be situated in the part of the molecule that is opposite to the NHAc group of GalNAc residue. In order to test this hypothesis we have synthesized a polymeric conjugate in such a way that de-N-acetylated A-trisaccharide is attached to a polymer via the nitrogen in position C-2 of the galactosamine residue. In this conjugate the supposed AB-epitope should be maximally accessible for antibodies from the solution, whereas the discrimination site of antigens A and B by the antibodies should be maximally hidden due to the close proximity of the polymer. Interaction with several anti-AB monoclonal antibodies revealed that a part of them really interacted with the synthetic AB-glycotope, thus confirming our hypothesis. Moreover, similar antibodies were revealed in the blood of healthy blood group 0 donors. Analysis of spatial models was performed in addition to identify the hydroxyl groups of Fuc, Galalpha, and Galbeta residues, which are particularly involved in the composition of the AB-glycotope.

Monomeric and multimeric blockers of selectins: comparison of in vitro and in vivo activity.

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO(3)Le(a)-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLe(x) is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO(3)Le(a)-PAA-sTyr.

Uncharged P-selectin blockers.

The blocking potency of P- and L-selectin was studied for certain small molecule mannosides and their polyacrylamide (PAA, 30 kDa) conjugates in comparison to SiaLe(x) and fucoidan. Two experimental systems were used: (1) solid phase static assay based on recombinant selectins, and (2) P-selectin dependent rat peritoneal inflammation. betaMan-SC6H4NO2- p was four times more potent P-selectin inhibitor as compared to SiaLe(x). Docking of this molecule onto the P-selectin carbohydrate-binding site demonstrated that a nitro group enabled an electrostatic interaction with residue Lys 84, while the phenyl ring and the CH2 at C-6 contacted the CH2 groups of the same Lys residue. In vivo, betaMan-SC6H4NO2- p blocked experimental inflammation better than SiaLe(x), but significantly lower than fucoidan. In vitro Man-polyacrylic acid conjugates appeared to be very potent inhibitors comparable to fucoidan, uncharged Man-PAA proved rather active, comparable to SiaLe(x)-PAA both in vitro, and in vivo, whereas mannan did not display any P-selectin blocking effect.

[Effects of mannose derivatives on development of selectin-dependent peritoneal inflammation in rats and mice]. / Vliianie proizvodnykh mannozy na razvitie selektinzavisimogo peritoneal'nogo vospaleniia u krys i myshei.

The inhibitory effect of mannose-containing oligosaccharides on model carbohydrate ligand interaction with E-, P- and L-selectins in vitro, as well as on the ability of these compounds to block the leukocyte extravasation in rat and mouse peritonitis in vivo was studied. The monomeric and polymeric compounds, 4-nitrophenylthiomannoside, phenylmannoside, conjugated with polyacrylic acid, and alpha-mannose, conjugated with polyacrylamide, inhibited the binding of the model ligand to P- and L-selectins (but not to E-selectin). Intravenous injection of these compounds was found to cause a dose-dependent reduction of neutrophil accumulation in rat peritoneum. The polysaccharide mannan was inactive in both types of experiments. The conjugate of phenylmannoside with polyacrylic acid was the most effective blocker as in vitro experiments, as well as in vivo. The inhibitory effect of subcutaneous injection of 4-nitrophenylthiomannoside on mouse peritonitis was demonstrated.

[Inhibitory activity of monomeric and polymeric selectin ligands]. / Ingibitornaia aktivnost' monomernykh i polimernykh ligandov selektinov.

The ability of tetrasaccharides (SiaLex, SiaLea, HSO3Lex), their conjugates with polyacrylamide (40 kDa), and several other monomeric and polymeric substances to block selectins has been compared with that of polysaccaride fucoidan. Two assay systems were used: one was constructed on the base of recombinant E-, P-, and L-selectins; the other was a rat model of peritoneal inflammation. IC50 values for the neoglycoconjugate SiaLea-PAA were 6, 40, and 85 microM with the recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-o-sulfate in addition to one of the above mentioned oligosaccharides, were the most potent synthetic blockers. Compared with the most potent of the known inhibitors, fucoidan, bi-ligand glycoconjugate HSO3Lea-PAA-sTyr, displayed in vitro similar activity in blocking L-selectin, while its activity towards P-selectin was ten times lower. All the synthetic polymers tested were able to inhibit neutrophil extravasation to inflammation site, acting in concentration about 10 mg/kg. Thus, the effect of SiaLex is considerably more effective in vivo than in vitro, whereas heavily charged fucoidan and bi-ligand neoglycoconjugate acted in converse manner.

[The tissue localization and secretion of mucin-D in Drosophila melanogaster]. / Tkanevaia lokalizatsiia i sekretsiia mutsina-D Drosophila melanogaster.

Glycoprotein with biochemical characteristics that allow us to classify it as a glycoprotein of mucin-type was isolated from cultured embryonic cells of Drosophila melanogaster. This is the first finding of mucin-type glycoprotein in insects. Using high-affinity monoclonal antibodies against a carbohydrate epitope, we demonstrated that the accumulation of this glycoprotein in the culture fluid of Drosophila cell line and cultured cells of other insects was inhibited by secretion inhibitors. 20-hydroxyecdysone, a hormone responsible for molting and metamorphosis in insects, inhibits the accumulation of this glycoprotein in the culture fluid, as well as in cells of Drosophila Dm cell line. In another cell line (Schneider-2), where there was practically no secretion of this glycoprotein, the hormone induced its increased accumulation in the cells. Mucin glycoproteins recognized by monoclonal antibodies have been found in embryos, imaginal disc, fat body, testicles, and ovaries, but not in salivary glands or muscles of Drosophila.

Insect mucin-type glycoprotein: immunodetection of the O-glycosylated epitope in Drosophila melanogaster cells and tissues.

A mucin-type glycoprotein (GP) from cultured embryonic cells of Drosophila melanogaster was isolated and used to raise monoclonal antibodies (MAbs). Epitope(s) recognized by MAbs were sensitive to the treatment by O-glycanase, which specifically cleaves off O-linked mucin-type Gal(beta 1,3)GalNAc disaccharide, representing the major part of the carbohydrate moiety of Drosophila GP. Using high-affinity MAbs against carbohydrate epitopes of the Drosophila mucin GP we demonstrated its accumulation in culture medium, as well as in cultured cells, which proved to be regulated by 20-hydroxyecdysone. Mucin GPs carrying Gal(beta 1,3)GalNAc disaccharide recognized by the MAbs were immunochemically localized in several Drosophila tissues of ectodermal, mesodermal and germ line origin, including epidermal and follicle cells capable of their secretion.

[Glycoproteins from Drosophila melanogaster cell culture contains O-bound carbohydrate chains of the Gal(beta1-3)GalNAc type]. / Glikoprotein iz kul'tury kletok Drosophila melanogaster soderzhit O-sviazannye uglevodnye tsepi tipa Gal(beta1-3)GalNAc.

The glycoprotein from cultured cells of D. melanogaster, also detected in various insect tissues as a component of the extracellular matrix, was characterized as a mucin-type glycoprotein not yet described in invertebrates. This glycoprotein with an apparent molecular mass of approximately 90 kDa contains about 40% of carbohydrates, largely represented mainly by GalNAc and Gal; its polypeptide moiety is enriched with Thr, Ser, Pro and Gly. An analysis of oligosaccharides liberated by treatment of the glycoprotein with alkaline NaBH4 or O-glycanase (endo-alpha-N-acetylgalactosaminidase) revealed Gal(beta 1-3)GalNAc as the major type of the sugar chains. About half of the Thr + Ser residues in the glycoprotein were estimated as O-glycosidically linked with the disaccharide units.

[Effects of monoclonal antibodies to bovine nerve growth factor on NGF-induced cell differentiation in culture]. / Vliianie monoklonal'nykh antitel k faktoru rosta nervov byka na FRN-indutsirovannuiu differentsirovku kletok v kul'ture.

Five Hybridoma clones producing monoclonal antibodies (MAT) to bovine nerve growth factor (NGF) were developed. The biological effects of antibodies were studied: the influence of MAT on neurit outgrowth induced by NGF in rat pheochromocytoma PC12 or spinal chicken ganglia was investigated. MAT fell into two groups. Two of them inhibited neurit induction by NGF, three others stimulated this process. The stimulation of the neurit outgrowth by MAT was observed at low concentration of NGF (3 ng/ml of culture medium). Mechanisms of antibodies effects are discussed.